title: Evaluation of LAMP assay using phenotypic tests and conventional PCR for detection of blaNDM-1 and blaKPC genes among carbapenem-resistant clinical Gram-negative isolates.
abstract: Carbapenem-resistant pathogens cause infections associated with significant morbidity and mortality. This study evaluates the use of the loop-mediated isothermal amplification (LAMP) assay for rapid and cost-effective detection of bla(NDM-1) and bla(KPC) genes among carbapenem-resistant Gram-negative bacteria in comparison with conventional PCR and existing phenotypic methods. A total of 60 carbapenem-resistant clinical isolates [Escherichia coli (15), Klebsiella pneumoniae (22), Acinetobacter baumannii (23)] were screened for the presence of carbapenemases (bla(KPC) and bla(NDM-1)) using phenotypic methods such as the modified Hodge test (MHT) and combined disc test (CDT) and molecular methods such as conventional PCR and LAMP assay. In all, 47/60 isolates (78.3%) were MHT positive while 48 isolates were positive by CDT [46.6% positive with EDTA, 30% with 3' aminophenylboronic acid (APB) plus EDTA and 1.6% with APB alone]. Isolates showing CDT positivity with EDTA or APB contained bla(NDM-1) and bla(KPC) genes, respectively. bla(NDM-1) was present as a lone gene in 28 isolates (46.7%) and present together with the bla(KPC) gene in 19 isolates (31.7%). Only one E. coli isolate had a lone bla(KPC) gene. The LAMP assay detected either or both bla(NDM-1) and bla(KPC) genes in four isolates that were missed by conventional PCR. Neither gene could be detected in 12 (20%) isolates. The LAMP assay has greater sensitivity, specificity and rapidity compared to the phenotypic methods and PCR for the detection of bla(NDM-1) and bla(KPC). With a turnaround time of only 2-3 h, the LAMP assay can be considered a point-of-care assay.
authors.affiliation: Nizams Institute of Medical Science, Hyderabad, Andhra Pradesh, India.
authors.forename: Rachana
authors.initials: R
authors.lastname: Solanki
authors.affiliation: Nizams Institute of Medical Science, Hyderabad, Andhra Pradesh, India.
authors.forename: Lavanya
authors.initials: L
authors.lastname: Vanjari
authors.affiliation: Nizams Institute of Medical Science, Hyderabad, Andhra Pradesh, India.
authors.forename: Nagapriyanka
authors.initials: N
authors.lastname: Ede
authors.affiliation: Jawaharlal Nehru Technological University, Hyderabad, Andhra Pradesh, India.
authors.forename: Akhila
authors.initials: A
authors.lastname: Gungi
authors.affiliation: Jawaharlal Nehru Technological University, Hyderabad, Andhra Pradesh, India.
authors.forename: Amarendranath
authors.initials: A
authors.lastname: Soory
authors.affiliation: Nizams Institute of Medical Science, Hyderabad, Andhra Pradesh, India.
authors.forename: Lakshmi
authors.initials: L
authors.lastname: Vemu
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001431:Bacteriological Techniques / Q000379:methods*; D015780:Carbapenems / Q000494:pharmacology; D004755:Enterobacteriaceae / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D004756:Enterobacteriaceae Infections / Q000382:microbiology*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D018440:beta-Lactam Resistance; D001618:beta-Lactamases / Q000235:genetics*
chemical_list: D000900:Anti-Bacterial Agents; D015780:Carbapenems; D001618:beta-Lactamases